Project Summary Nucleosomes are the elementary and repeating building blocks of chromatin, comprised of a histone octamer wrapped in 147 bp DNA. Histone post-translational modifications (PTMs) play a dynamic role in controlling gene expression and other essential cellular functions. These PTMs form a systems-level signaling network (i.e., the histone code) that recruits specific epigenetic effector proteins to transduce various downstream signaling cascades. So-called `reader' proteins are compelling candidates for therapeutic manipulation of gene expression in multiple diseases, including cancer, neurodegeneration, and other conditions. However, high-throughput methods are not available to comprehensively evaluate the interaction between physiologically-relevant PTM states and their regulatory protein partners. In addition, PTM targets are often studied on a synthetic peptide backbone, a substrate that poorly models in vivo chromatin structure. To address these issues, EpiCypher is developing a barcoded nucleosome platform for high-throughput PTM- protein interaction studies. Toward this goal, our group has developed proprietary methods for the commercial production of recombinant histones customized with a broad range of PTMs (termed `designer nucleosomes' or `dNucs'). In Phase I, we synthesized dNuc substrates carrying single and combinatorial PTMs, to establish EpiCodeTM, a powerful barcoding technology for multiplexed protein-PTM interaction studies. The innovation of this technology is the ability to screen potentially hundreds of protein-nucleosomal PTM combinations in a single experiment. EpiCodeTM employs uniquely modified dNucs identified by barcodes appended to their DNA assembly sequence. DNA barcoding is central to our approach as it allows multiplexed binding events to be deconvolved using qPCR. EpiCodeTM allows specific proteins-of-interest to be combined with a PTM- customized dNuc library to identify new interactions. In Phase II, our first goal is to generate an expanded barcoded dNuc library targeting the combinatorial impact of histone acetylation and methylation, two highly prevalent PTM families with significant disease- relevance (Aim 1). We will validate our expanded barcoded dNuc library by performing multiplexed binding assays using a collection of readers that interact with PTMs represented in the library (Aim 2). As part of this validation, we will establish protocols compatible with next-generation sequencing (NGS; used in place of qPCR) for streamlined deconvolution of multiplexed barcodes. In Aim 3, we will develop simplified workflows that utilize plate-based assay designs, providing high-throughput assays for major end users interested in using this platform for large discovery projects (i.e., pharmaceutical companies). Finally, several external laboratory sites will validate our platform with a goal of refining protocols (to minimize/characterize variability) and demonstrating its utility as a tool to identify novel protein-PTM interactions. As a powerful new tool to decode chromatin signaling, EpiCodeTM will be of broad interest to both pharmaceutical and basic researchers.